Necdin promotes ubiquitin-dependent degradation of PIAS1 SUMO E3 ligase.

Ibrahim Gur,Kazushiro Fujiwara,Kazuaki Yoshikawa,Koichi Hasegawa,Thomas G Hofmann

doi:10.1371/journal.pone.0099503

Ibrahim Gur, Kazushiro Fujiwara + Show 3 more

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https://doi.org/10.1371/journal.pone.0099503

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Journal: PloS one	Publication Date: Jun 9, 2014
Citations: 14	License type: CC BY 4.0

Affiliation: Protein Research Foundation, Osaka University

Abstract

Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation.

Highlights

Necdin was originally identified as a hypothetical protein encoded by a gene transcript expressed in neurally differentiated P19 embryonal carcinoma cells [1]
We analyzed the interactions of typical SUMO E3 ligases including Nsmce2, Cbx4 and PIAS1 with MAGE proteins including necdin, MAGEA1, MAGED1, MAGEF1, necdin-like 2 and MAGEL2 by co-immunoprecipitation assay
We found that Nsmce1 interacted with MAGEA1, necdin-like 2 and MAGEF1, but not with necdin, MAGED1 or MAGEL2 (Fig. 1A)

Summary

Introduction

Necdin was originally identified as a hypothetical protein encoded by a gene transcript expressed in neurally differentiated P19 embryonal carcinoma cells [1]. Necdin interacts with many regulatory proteins including E2F family proteins and p53 [7,8,9,10,11] and promote survival and differentiation of neurons and neural stem/progenitor cells [5,6,10,11,12,13]. MAGE genes per genome [14,15], whereas only a single MAGE gene has been identified in invertebrates such as the fruit fly (Drosophila melanogaster) [16] and non-mammalian vertebrates such as the zebrafish (Danio rerio) [17] and chicken (Gallus gallus) [18]. Nonmammalian MAGE genes in the fruit fly [24,25], zebrafish [17] and chicken [18] are expressed during neurogenesis, suggesting that MAGE family genes are involved in neuronal development

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